Brit. Heart ., 1968, 30, 265. Electron Microscopy of the Heart in a Case of Primary Cardiac Amyloidosis E. M. HUSBAND AND R. LANNIGAN From the Department of Pathology, University of Birmingham Although electron microscope studies of the ultrastructure of amyloid and its location in kidney and other organs (Cohen and Calkins, 1959; Heefner and Sorenson, 1962; Gueft and Ghidoni, 1963) have been carried out, there are no reports of the electron microscopy of amyloid in the heart. This communication describes the electron micro- scope findings in the heart from a case of primary amyloidosis. MATERIAL AND METHOD The patient was a male Pakistani who died at the age of 35 from renal and cardiac amyloidosis. The duration of the illness was short, the first symptoms appearing only 3 months before death. The initial manifestations of the disease were those of the nephrotic syndrome, but intrac- table congestive cardiac failure developed shortly after- wards and was considered at necropsy to be the cause of death. Post-mortem examination was carried out 16 hours after death. Only the relevant necropsy findings will be described. There was moderate oedema of both lower limbs. Bilateral serous pleural effusions were present and there was a 300 ml. pericardial effusion. The liver and spleen were both enlarged (2000 g. and 395 g., respectively). Both showed severe passive venous congestion. The heart was enlarged (weight 550 g.), with uniform en- largement of all chambers. The myocardium was firmer than normal and appeared waxy. The tongue was enlarged and showed indentations from the teeth around the periphery. The thyroid gland was uniformly en- larged and weighed 50 g. The kidneys were equally enlarged (combined weight 425 g.); the parenchyma was paler and firmer than normal and waxy in appearance. For light micropscopy thin slices of cardiac and other tissues were fixed in 10 per cent formol-saline and embedded in paraffin wax. Sections were stained with haematoxylin and eosin, haematoxylin and van Gieson, periodic acid-Schiff, Congo-red, and crystal violet. For electron microscopy formalin-fixed cardiac tissue was cut into 1 mm. 3 blocks, washed in distilled water for Received March 20, 1967. 265 48 hours, refixed in 1 per cent buffered osmium tetroxide for 1 hour, and embedded in araldite. Thin sections on carbon-coated grids were stained with lead citrate and examined in an AEI EM6B electron microscope at an accelerating voltage of 60 kV. OBSERVATIONS Light microscopy showed extensive deposits of amyloid in heart (Fig. 1), thyroid, kidney, and tongue. The cardiac deposits were present in the interstitium of both atria and ventricles. Electron Microscopy. Although the cardiac tissue was 16 hours post mortem before formalin-fixation, some cytological detail was recognizable; the myo- fibrils were well preserved, and Z bands and even the thick and thin filaments were clearly seen. The cell membrane, however, was partly disrupted and the mitochondria were swollen and their cristae fragmented. Amyloid deposits were present in relation to the basement membranes of cardiac muscle cells (Fig. 2a), around capillaries (Fig. 2b), and in the con- nective tissues (Fig. 2c). At low magnifications the deposits appeared granular but at higher mag- nifications they were seen to consist of fine fibrils. Some of the fibrils were disposed haphazardly, others were arranged in bundles (Fig. 2d). The fibrils varied in width from 140A-450A; those of greatest diameter appeared to be present in the bundles. In some areas there was a suggestion of regular beading along the fibrils. In the connective tissue amyloid fibrils and col- lagen fibrils were both present. It was easy to distinguish the much larger mature collagen fibres with their characteristic periodicity (600A) from the more slender amyloid fibrils. The mature collagen fibrils showed no loss of periodicity. Extensive deposits of amyloid were present between the muscle cells which were reduced in size. Large numbers of mitochondria were on February 7, 2021 by guest. Protected by copyright. http://heart.bmj.com/ Br Heart J: first published as 10.1136/hrt.30.2.265 on 1 March 1968. Downloaded from
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Brit. Heart ., 1968, 30, 265.
Electron Microscopy of the Heart in a Caseof Primary Cardiac Amyloidosis
E. M. HUSBAND AND R. LANNIGAN
From the Department of Pathology, University of Birmingham
Although electron microscope studies of theultrastructure of amyloid and its location in kidneyand other organs (Cohen and Calkins, 1959;Heefner and Sorenson, 1962; Gueft and Ghidoni,1963) have been carried out, there are no reports ofthe electron microscopy of amyloid in the heart.This communication describes the electron micro-scope findings in the heart from a case of primaryamyloidosis.
MATERIAL AND METHODThe patient was a male Pakistani who died at the age of
35 from renal and cardiac amyloidosis. The duration ofthe illness was short, the first symptoms appearing only 3months before death. The initial manifestations of thedisease were those of the nephrotic syndrome, but intrac-table congestive cardiac failure developed shortly after-wards and was considered at necropsy to be the cause ofdeath.Post-mortem examination was carried out 16 hours
after death. Only the relevant necropsy findings will bedescribed.There was moderate oedema of both lower limbs.
Bilateral serous pleural effusions were present and therewas a 300 ml. pericardial effusion. The liver and spleenwere both enlarged (2000 g. and 395 g., respectively).Both showed severe passive venous congestion. Theheart was enlarged (weight 550 g.), with uniform en-
largement of all chambers. The myocardium was firmerthan normal and appeared waxy. The tongue was
enlarged and showed indentations from the teeth aroundthe periphery. The thyroid gland was uniformly en-
larged and weighed 50 g. The kidneys were equallyenlarged (combined weight 425 g.); the parenchyma was
paler and firmer than normal and waxy in appearance.For light micropscopy thin slices of cardiac and other
tissues were fixed in 10 per cent formol-saline andembedded in paraffin wax. Sections were stained withhaematoxylin and eosin, haematoxylin and van Gieson,periodic acid-Schiff, Congo-red, and crystal violet.
For electron microscopy formalin-fixed cardiac tissuewas cut into 1 mm. 3 blocks, washed in distilled water for
Received March 20, 1967.265
48 hours, refixed in 1 per cent buffered osmium tetroxidefor 1 hour, and embedded in araldite. Thin sections oncarbon-coated grids were stained with lead citrate andexamined in an AEI EM6B electron microscope at anaccelerating voltage of 60 kV.
OBSERVATIONSLight microscopy showed extensive deposits of
amyloid in heart (Fig. 1), thyroid, kidney, andtongue. The cardiac deposits were present in theinterstitium of both atria and ventricles.
Electron Microscopy. Although the cardiac tissuewas 16 hours post mortem before formalin-fixation,some cytological detail was recognizable; the myo-fibrils were well preserved, and Z bands and eventhe thick and thin filaments were clearly seen. Thecell membrane, however, was partly disrupted andthe mitochondria were swollen and their cristaefragmented.Amyloid deposits were present in relation to the
basement membranes of cardiac muscle cells (Fig.2a), around capillaries (Fig. 2b), and in the con-nective tissues (Fig. 2c). At low magnifications thedeposits appeared granular but at higher mag-nifications they were seen to consist of fine fibrils.Some of the fibrils were disposed haphazardly,others were arranged in bundles (Fig. 2d). Thefibrils varied in width from 140A-450A; those ofgreatest diameter appeared to be present in thebundles. In some areas there was a suggestion ofregular beading along the fibrils.
In the connective tissue amyloid fibrils and col-lagen fibrils were both present. It was easy todistinguish the much larger mature collagen fibreswith their characteristic periodicity (600A) from themore slender amyloid fibrils. The mature collagenfibrils showed no loss of periodicity.
Extensive deposits of amyloid were presentbetween the muscle cells which were reduced insize. Large numbers of mitochondria were
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FIG. 1.-Myocardium showing extensive amyloid deposits apparently replacing muscle fibres and extendingbetween existing fibres. (Crystal violet. x 350.)
present, however, and the myofibrils were easilyrecognizable. A few cells contained deposits oflipofuscin. The remains of the cell membrane wereseen in some areas but in other areas it had disap-peared and was replaced by masses of amyloidwhich formed a thick mass around the cell. Amy-loid deposition inside the myocardial cells was notseen.Only occasional capillaries were recognized, and
where extensive deposits of amyloid were present inthe connective tissue there seemed to be a reductionin the number of capillaries. Masses of amyloidsurrounded those which were seen and in some areasthe capillary basement membrane appeared to bereplaced by amyloid. Occasionally the appear-ances suggested that amyloid was in direct contactwith the capillary lumen.A few cells of the macrophage type were seen in
the connective tissues but no intracellular amyloidwas observed.
DISCUSSIONAmyloid deposits in human primary and secon-
dary amyloidosis and in experimentally inducedamyloidosis, which have been studied in the electronmicroscope, have all been seen to have a finefibrillary structure. A similar fibrillary appearancewas recognized in the deposits in the heart in thiscase.The width of the individual fibrils in the cardiac
deposits varied between 140A-450A. There was
also considerable variation (75A-300A) in the widthof the fibrils in previous reports of the ultrastructureof amyloid (Cohen, Weiss, and Calkins, 1960; Boere,Ruinen, and Scholten, 1965). Gueft and Ghidoni(1963) observed that some fibrils appeared to becomposed of two filaments and that cross-striationsappeared to be present. Shirahama and Cohen in1965 demonstrated by electron microscopy of nega-tively stained amyloid that each fibril was composedof from 1-8 filaments, each of which was 75A indiameter. This provides an explanation for thevariation in the width of the fibrils.The amyloid in this case was situated in the inter-
cellular space in close relation to the plasma mem-brane of the muscle cells and the basementmembrane of the capillaries. In some areas thecapillary basement membrane appeared to havedisappeared and amyloid seemed to be in contactwith the lumen of the vessel. Although this mayhave been an artefact or possibly the result of auto-lytic change, it has been observed that basementmembrane is well preserved in necropsy material(Ashworth and Stembridge, 1964). Moreoverother authors have described a similar appearancein fresh osmium-tetroxide fixed material (Cohen etal., 1960).
In many areas mature collagen fibres were presentin close relation to amyloid deposits. The collagendid not appear to be taking part in the formation ofthe deposit. Evidence in support of this is the factthat isolated amyloid fibrils remain unchanged after
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Electron Microscopy of the Heart in a Case of Primary Cardiac Amyloidosis
FIG. 2a.-Oblique section of muscle cell showing preserved myofibrils (My) and intercalated disc (ID)surrounded by amyloid (A). The basement membrane (BM) can be recognized in places. The mito-
chondria (MT) are swollen and show fragmentation of the cristae. ( x 3700.)FIG. 2b.-Capillary surrounded by amyloid (A). The nuclei of two pericytes are prominent (N). The
basement membrane (arrow) can be seen at some areas. ( x 2700.)FIG. 2c.-Collagen fibrils (C) with well-defined characteristic periodicity together with amyloid fibrils (A) in
connective tissue. (x 33,000.)FIG. 2d.-Amyloid fibrils, parly arranged haphazardly, and partly arranged in bundles. (x 27,000.)
treatment with collagenase and hyaluronidase(Cohen and Calkins, 1964). The number of capil-laries in the intercellular zones appeared to bereduced, a factor that may have been important inthe production of muscle cell atrophy. The intrac-table congestive cardiac failure which is the com-monest manifestation of cardiac amyloidosis(Lindsay, 1946; Benson and Smith, 1956) may bethe result of myocardial atrophy and interferencewith cellular nutrition. It may however be the
result of mechanical interference with diastolic ex-pansion and filling of the heart due to the presence oflarge amounts of relatively inelastic amyloid mat-erial. This is supported by cardiac catheterizationstudies which in cardiac amyloidosis give resultssimilar to constrictive pericarditis (Gunnar et al.,1955).Although cytoplasmic changes presumably occur
in muscle cells in advanced amyloidosis, any suchchanges in this case were obscured by autolysis.
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Clumping of nuclear chromatin, swelling of mito-chondria, and disruption of the cell membrane areearly autolytic changes (Ashworth and Stembridge,1964) and these were the only striking cellularchanges seen in the myocardial cells. Althoughfresh osmium-fixed tissue is essential for cytologicaldetail, certain components of connective tissue arewell preserved in necropsy material (Lannigan andZaki, 1965; Lehner, Nunn, and Pearse, 1966), anduseful information may be obtained by electronmicroscopy of such material.
SUMMARYThe electron microscope appearances of post-
mortem cardiac tissue from a case of primary car-diac amyloidosis are described. The mechanismswhich may be responsible for congestive cardiacfailure in myocardial amyloidosis are briefly dis-cussed.
REFERENCESAshworth, C. T., and Stembridge, V. A. (1964). Utility of
formalin fixed surgical and autopsy specimens for elec-tron microscopy. Amer. j. clin. Path., 42, 466.
Benson, R., and Smith, J. F. (1956). Cardiac amyloidosis.Brit. Heart J., 18, 529.
Boere, N., Ruinen, L., and Scholten, J. H. (1965). Electronmicroscopic studies on the fibrillar component ofhumansplenic amyloid. J. Lab. clin. Med., 66, 943.
Cohen, A. S., and Calkins, E. (1959). Electron microscopicobservations on a fibrous component in amyloid ofdiverse origins. Nature (Lond.), 183, 1202.,and - (1964). The isolation of amyloid fibrils and astudy of the effect of collagenase and hyaluronidase.J7. Cell Biol., 21, 481.
, Weiss, L., and Calkins, E. (1960). Electron micro-scopic observations of the spleen during the induction ofexperimental amyloidosis in the rabbit. Amer. J. Path.,37, 413.
Gueft, B., and Ghidoni, J. J. (1963). The site of formationand ultra-structure of amyloid. Amer.J. Path., 43, 837.
Gunnar, R. M., Dillon, R. F., Wallyn, R. J., and Elisberg, E. I.(1955). The physiologic and clinical similarity betweenprimary amyloid of the heart and constrictive peri-carditis. Circulation, 12, 827.
Heefner, W. A., and Sorenson, G. D. (1962). Experimentalamyloidosis. Light and electron microscope observa-tions of spleen and lymph nodes. Lab. Invest., 11, 585.
Lannigan, R., and Zaki, S. (1965). Electron microscopeappearances of a fibrin-staining component in an Aschoffnodule from acute rheumatic fever. Nature (Lond.),206, 106.
Lehner, T., Nunn, R. E., and Pearse, A. G. E. (1966).Electron microscopy of paraffin-embedded material inamyloidosis. J. Path. Bact., 91, 297.
Lindsay, S. (1946). The heart in primary systemic amy-loidosis. Amer. Heart_J., 32, 419.
Shirahama, T., and Cohen, A. S. (1965). Structure of amy-loid fibrils after negative staining and high-resolutionelectron microscopy. Nature (Lond.), 206, 737.
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